The goal of this project is to characterize mechanisms by which cardiolipin (CL) binding affects the structure and function of three mitochondrial electron transport complexes: cytochrome c oxidase, cytochrome bc1, and NADH dehydrogenase (Complex I). Our understanding of CL's role within each complex is at different stages. The purpose of our proposed experiments is to determine the function of CL within each complex and to define a possible universal binding motif. Cardiolipin binding to cytochrome c oxidase is well defined and the binding sites have been localized within the enzyme. The goal with cytochrome c oxidase is to determine whether CL facilitates proton entry into the binuclear center of cytochrome c oxidase and to identify the functional mechanism. Cardiolipin binding to cytochrome bc1 is less well defined. CL is tightly bound to cyt bc1 and is critically required for structural stability and maintenance of activity. However, the binding sites within cyt bc1 isolated from higher eukaryotes are not defined. We propose to map the 4-6 binding sites within Cyt bc1 to determine the subunits and amino acid sequences participating in CL binding. Identified binding sites will be compared with the known PL binding sites within yeast cyt bc1 and bovine heart cytochrome c oxidase. Phospholipid binding to Complex I has not been defined. We have found that 2 CL and 2 PE are tightly associated with the purified enzyme, but their structural and functional importance has not been established. We will determine if CL is an essential component of Complex I and map its location within the complex. These specific aims are logical extensions of our previous studies. They take advantage of experimental approaches developed during past funding periods, i.e., 1) synthesis of arylazido-CL photolabels;2) analysis of subunit content using sensitive HPLC and mass spectrometry methods;and 3) selective dissociation of subunits using mild structural perturbants. With our cardiolipin analogues and experimental approaches, we are in a unique position to define the mechanisms by which tightly bound CL influences the function and/or structure of three key enzymes within the mitochondrial inner membrane.Public Health Relevance. Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Oxidative damage or age related loss of cardiolipin is associated with apoptosis and a number of diseases, e.g., ischemia/reperfusion injury and age-related degeneration. In this research project we will determine the cardiolipin requirements of three inner membrane electron transfer complexes and map the cardiolipin binding sites within each complex. Successful completion of this project will determine the absolute requirement for cardiolipin and help us understand its pathophysiological importance.